ENTAMEBOSIS

______________________________________
Entamoeba histolytica

Causal Agent:
Several protozoan species in the genus Entamoeba infect humans, but not all of
them are associated with disease. Entamoeba histolytica is well recognized as
a pathogenic ameba, associated with intestinal and extraintestinal infections.
The other species are important because they may be confused with E.
histolytica in diagnostic investigations.

Life Cycle:

Cysts and trophozoites are passed in feces .  Cysts are typically found in
formed stool, whereas trophozoites are typically found in diarrheal stool. 
Infection by Entamoeba histolytica occurs by ingestion of mature cysts in

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 fecally contaminated food, water, or hands.  Excystation occurs in the
small intestine and trophozoites are released, which migrate to the large
intestine.  The trophozoites multiply by binary fission and produce cysts ,
and both stages are passed in the feces .  Because of the protection
conferred by their walls, the cysts can survive days to weeks in the external
environment and are responsible for transmission.  Trophozoites passed in
the stool are rapidly destroyed once outside the body, and if ingested would
not survive exposure to the gastric environment.  In many cases, the
trophozoites remain confined to the intestinal lumen ( : noninvasive
infection) of individuals who are asymptomatic carriers, passing cysts in their
stool.  In some patients the trophozoites invade the intestinal mucosa ( :
intestinal disease), or, through the bloodstream, extraintestinal sites such as
the liver, brain, and lungs ( : extraintestinal disease), with resultant
pathologic manifestations.  It has been established that the invasive and
noninvasive forms represent two separate species, respectively E.
histolytica and E. dispar.  These two species are morphologically
indistinguishable unless E. histolytica is observed with ingested red blood
cells (erythrophagocystosis).  Transmission can also occur through
exposure to fecal matter during sexual contact (in which case not only cysts,
but also trophozoites could prove infective).

Geographic Distribution:
Worldwide, with higher incidence of amebiasis in developing countries. In
industrialized countries, risk groups include male homosexuals, travelers and
recent immigrants, and institutionalized populations.

Clinical Features:
A wide spectrum, from asymptomatic infection ("luminal amebiasis"), to invasive
intestinal amebiasis (dysentery, colitis, appendicitis, toxic megacolon,
amebomas), to invasive extraintestinal amebiasis (liver abscess, peritonitis,
pleuropulmonary abscess, cutaneous and genital amebic lesions).

Laboratory Diagnosis:
Entamoeba histolytica must be differentiated from other intestinal protozoa
including: E. coli, E. hartmanni, E. gingivalis, Endolimax nana, and Iodamoeba
buetschlii (the nonpathogenic amebas); Dientamoeba fragilis (which is a
flagellate not an ameba); and the possibly pathogenic Entamoeba polecki.
Differentiation is possible, but not always easy, based on morphologic
characteristics of the cysts and trophozoites. The nonpathogenic Entamoeba
dispar, however, is morphologically identical to E. histolytica, and differentiation
must be based on isoenzymatic or immunologic analysis.
Molecular methods are also useful in distinguishing between E. histolytica and
E. dispar and can also be used to identify E. polecki. Microscopic identification
of cysts and trophozoites in the stool is the common method for diagnosing E.
histolytica.

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This can be accomplished using:
Fresh stool: wet mounts and permanently stained preparations (e.g., trichrome).
Concentrates from fresh stool: wet mounts, with or without iodine stain, and
permanently stained preparations (e.g., trichrome).
Concentration procedures, however, are not useful for demonstrating
trophozoites.
In addition, E. histolytica trophozoites can also be identified in aspirates or
biopsy samples obtained during colonoscopy or surgery.

Diagnostic findings:
 Microscopy.
 Immunodiagnosis.
 Molecular methods for discriminating between E. histolytica and E. dispar

Treatment:
For asymptomatic infections, iodoquinol, paromomycin, or diloxanide furoate
(not commercially available in the U.S.) are the drugs of choice. For
symptomatic intestinal disease, or extraintestinal, infections (e.g., hepatic
abscess), the drugs of choice are metronidazole or tinidazole, immediately
followed by treatment with iodoquinol, paromomycin, or diloxanide furoate.

Microscopy
Cysts
Mature Entamoeba histolytica / Entamoeba dispar cysts have 4 nuclei that
characteristically have centrally located karyosomes and fine, uniformly
distributed peripheral chromatin. Cysts usually measure 12 to 15 µm.

A B

A: Line drawing of an E. histolytica / E. dispar cyst.

B: E. histolytica / E. dispar in a concentrated wet mount stained with iodine. 
The cysts are usually spherical and often have a halo. The cyst in A appears
uninucleate.

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 C D

C, D: E. histolytica / E. dispar cysts stained with trichrome.  Two to three nuclei

are visible in the focal plane (black arrows), and the cysts contain chromatoid
bodies with typically blunted ends (red arrows).  The chromatoid body in C is
particularly well demonstrated.

Trophozoites
Entamoeba histolytica / Entamoeba dispar trophozoites have a single nucleus,
which have a centrally placed karyosome and uniformly distributed peripheral
chromatin. This typical appearance of the nucleus is not always observed as
some trophozoites can have nuclei with an eccentric karyosome and unevenly
distributed peripheral chromatin. The cytoplasm has a granular or "ground-
glass" appearance. E. histolytica / E. dispar trophozoites usually measure 15 to
20 µm (range 10 to 60 µm) tending to be more elongated in diarrheal stool.
Erythrophagocytosis (ingestion of red blood cells by the parasite) is the only
morphologic characteristic that can be used to differentiate E. histolytica from
the nonpathogenic E. dispar. However, erthrophagocytosis is not typically
observed on stained smears of E. histolytica.

E F

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E: Line drawing of an E. histolytica / E. dispar trophozoite.
F: E. histolytica / E. dispar trophozoite in a direct wet mount stained with iodine.

G H

G: E. histolytica / E. dispar trophozoite, measuring approximately 16.7 µm,

stained with trichrome. 
H: E. histolytica trophozoite.  The specimen was preserved in poly-vinyl alcohol
(PVA) and stained in trichrome.  PCR was performed on this specimen to
differentiate between E. histolytica and E. dispar.

Trophozoite of E. histolytica with

ingested erythrocytes stained with
trichrome.
The ingested erythrocyte appears as a
dark inclusion.
Erythrophagocytosis is the only
characteristic that can be used to
differentiate morphologically E. histolytica
from the nonpathogenic E. dispar.

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Immunodiagnosis

Antibody Detection
Enzyme immunoassay (EIA) kits for Entamoeba histolytica antibody detection
as well as EIA kits for antigen detection are commercially available in the United
States. Antibody detection is most useful in patients with extraintestinal disease
(i.e., amebic liver abscess) when organisms are not generally found on stool
examination. Antigen detection may be useful as an adjunct to microscopic
diagnosis in detecting parasites and can distinguish between pathogenic and
nonpathogenic infections.

The indirect hemagglutination (IHA) test has been replaced by commercially

available EIA test kits for routine serodiagnosis of amebiasis. Antigen consists
of a crude soluble extract of axenically cultured organisms. The EIA test
detects antibody specific for E. histolytica in approximately 95% of patients with
extraintestinal amebiasis, 70% of patients with active intestinal infection, and
10% of asymptomatic persons who are passing cysts of E. histolytica. If
antibodies are not detectable in patients with an acute presentation of
suspected amebic liver abscess, a second specimen should be drawn 7-10
days later. If the second specimen does not show seroconversion, other agents
should be considered.

Detectable E. histolytica-specific antibodies may persist for years after

successful treatment, so the presence of antibodies does not necessarily
indicate acute or current infection. Specificity is 95% or higher: false-positive
reactions rarely occur. Although the immunodiffusion test is as specific, it is
slightly less sensitive than the IHA and EIA and requires a minimum of 24 hours
to obtain a result, in contrast to 2 hours required for the IHA or EIA tests.
However, the simplicity of the procedure makes it ideal for the laboratory that
has only an occasional specimen to test.

The IHA and EIA tests are more suitable for laboratories that have frequent
requests for amebiasis serology. Although detection of IgM antibodies specific
for E. histolytica has been reported, sensitivity is only about 64% in patients with
current invasive disease. Several commercial EIA kits for antibody detection
are available in the United States.

Antigen Detection
Antigen detection may be useful as an adjunct to microscopic diagnosis in
detecting parasites and to distinguish between pathogenic and nonpathogenic
infections. Recent studies indicate improved sensitivity and specificity of fecal
antigen assays with the use of monoclonal antibodies which can distinguish
between E. histolytica and E. dispar infections. At least one commercial kit is
available which detects only pathogenic E. histolytica infection in stool; several
kits are available which detect E. histolytica antigens in stool but do not exclude
E. dispar infections.

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Molecular diagnosis
Conventional PCR

In reference diagnosis laboratories, molecular analysis by PCR-based assays is

the method of choice for discriminating between the pathogenic species (E.
histolytica) and the nonpathogenic species (E. dispar).

Agarose gel (2%) analysis of a PCR diagnostic test for differentiation between
E. histolytica and E. dispar.

Lanes 1 - 4:
Amplification with the Psp3/Psp51 PCR primer pair specific for E. histolytica.
Diagnostic band size - 876 bp.
Lanes 6 - 9:
Amplification with the NPsp3/NPsp51 PCR primer pair specific for E. dispar.
Diagnostic band size - 876 bp.
Lanes 1 and 6:
E. histolytica 200:NIH, zymodeme II (positive control for E. histolytica).
Lanes 2 and 7:
E. dispar 351:IMMiT, zymodeme I (positive control for E. dispar).
Lanes 3 and 8:
Specimen from a patient with a liver abscess (positive with E. histolytica primers
and negative with E. dispar primers). E. histolytica 333:IMMiT, zymodeme XIV.
Lanes 4 and 9:
Specimen from an asymptomatic patient (positive with E. dispar primers and
negative with E. histolytica primers). E. dispar 389:IMMiT, zymodeme I.
Lane 5:
Molecular base pair standard, 100-bp ladder (from 600 to 1,000 bp).
Figure A contributed by Assist. Prof. Przemyslaw Myjak, Ph.D., Institute of
Maritime and Tropical Medicine, Gdynia, Poland.

Real-Time PCR
A TaqMan real-time PCR approach has been validated at CDC and is used for
differential laboratory diagnosis of amebiasis. The assay targets the 18S rRNA
gene with species-specific TaqMan probes in a duplex format, making it
possible to detect both species in the same reaction vessel.

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Other intestinal amebae that may be
mistaken for E. histolytica / E.dispar
______________________________________
Endolimax nana
Entamoeba coli
Entamoeba hartmanni
Entamoeba polecki
Iodamoeba buetschlii

Endolimax nana

Mature cysts of Endolimax nana have 4 nuclei with large, blot-like karyosomes
and do not have chromatoid bodies.  The cysts measure 6 to 8 µm (range 5 to
10 µm).

A B

A: Line drawing of an E. nana cyst.

B: E. nana cyst in a direct wet mount, under differential interference contrast (DIC)
microscopy.

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 E. nana cyst in a direct wet mount
stained with iodine.

E. nana cyst stained with

trichrome.

A B

Endolimax nana trophozoites each have one nucleus with a characteristically

large, irregularly shaped, blot-like karyosome.  Their nucleus has no peripheral

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chromatin.  Their cytoplasm is granular and vacuolated.  The trophozoites
measure usually 8 to 10 µm (range 6 to 12 µm).

A: Line drawing of an E. nana trophozoite. B: Trophozoite stained (trichrome).

C D

C, D: E. nana trophozoites stained with trichrome.

E. nana trophozoite stained with

trichrome and measuring about 9
µm. 

Entamoeba coli

Mature Entamoeba coli cysts typically have 8 nuclei, and measure usually 15
to 25 µm (range 10 to 35 µm).  E. coli is the only species of Entamoeba with
more than four nuclei in the cyst stage.  Chromatoid bodies are seen less
frequently than in E. histolytica.  When present they are usually splinter like with

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pointed ends and thus different from the chromatoid bodies of E. histolytica,
which have rounded ends.

A B

A: Line drawing of an E. coli cyst. B: E. coli cyst in a concentrated wet mount.

C D

C:  E. coli cyst in a concentrated wet mount stained with iodine.  The cyst in
C shows 5 nuclei in this focal plane. D:  E. coli cyst in a formalin concentrated
wet mount stained with iodine.

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 E F

E, F: E. coli cysts in a formalin concentrated wet mount stained with iodine.

A B

A, B: E. coli cysts stained with trichrome.  The cyst in A is an immature,

binucleate form, frequently seen with E. coli, in which a large glycogen vacuole
pushes the nuclei to opposite sides.

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 C D

C, D: E. coli cysts stained with trichrome.

The trophozoites of Entamoeba coli measure usually 20 to 25 µm, but they can be elongated
and reach up to 50 µm.  The trophozoites each have one nucleus with a characteristically large,
eccentric karyosome and coarse, irregular peripheral chromatin.  The cytoplasm is coarse and
vacuolated ("dirty" cytoplasm).

A B

A: Line drawing of an E. coli trophozoite.

B: E. coli trophozoite stained with trichrome.  Occasionally the cytoplasm
contains ingested bacteria (as seen in B), yeasts, or other materials.

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 C D

C, D: E. coli trophozoites stained with trichrome.

E F

E: An enlongated trophozoite of E. coli stained with trichrome.  The cytoplasm

of this trophozoite is "dirty."
F: E. coli trophozoite, measuring over 30 µm.  Note the pseudopodia extending
from the ameba. 

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Entamoeba hartmanni

Mature cysts of Entamoeba hartmanni have 4 nuclei and elongated chromatoid

bodies with rounded ends.  Cysts of E. hartmanni measure usually 6 to 8 µm
(range 5 to 10 µm) and are smaller than those of E. histolytica (10 to 20 µm).

A B

A: Line drawing of an E. hartmanni cyst. B: E. hartmanni cyst in a wet mount

stained with iodine.

Entamoeba hartmanni is often called a "small E. histolytica" because these two

species share many morphological characteristics, except their size. 
Trophozoites of E. hartmanni measure usually 8 to 10 µm (range 5 to 12 µm)
and are smaller than those of E. histolytica (10 to 60 µm).  The trophozoites of
E. hartmanni have one nucleus with fine peripheral chromatin and a small, often
centrally located karyosome.  The cytoplasm is finely granular.

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 A B

A: Line drawing of an E. hartmanni trophozoite.

B: E. hartmanni trophozoite stained with trichrome and measuring 8.1 µm. 

C D

C, D:  E. hartmanni trophozoites stained with trichrome.  Note that in C, the

trophozoite has ingested a yeast not an erythrocyte.  Ingestion of erythrocytes is
pathognomonic of E. histolytica.

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 E. hartmanni trophozoite stained
with trichrome.  A Blastocystis
hominis cyst-like form is in the
upper right corner of the image.

Entamoeba polecki

Cysts have one nucleus, or rarely two nuclei, with a small, usually eccentric
karyosome (which can also be rather pleomorphic).  Their cytoplasm contains
small inclusions and an "inclusion mass", which stains only weakly with iodine. 
The cysts measure usually 11 to 15 µm (range 9 to 18 µm) and their shape varies
from spherical to oval.

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 A B

A: Line drawing of an E. polecki cyst.

B: E. polecki cyst in a concentrated wet mount stained with iodine.

A B

A, B: E. polecki cysts stained with trichrome.  Note the large number of red

chromatoid bodies; these may be variable in shape.

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 C D

E F

C, D, E, F: E. polecki cysts stained with trichrome.

Each Entamoeba polecki trophozoites has one nucleus that usually has small,
discrete karyosomal chromatin and evenly distributed peripheral chromatin. 
Their cytoplasm is coarsely granular, vacuolated and can contain bacteria and
yeasts.  The trophozoites usually measure 15 to 20 µm (range 10 to 25 µm).

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 Line drawing of an E.
polecki trophozoite.

B C

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 D E

B, C, D, E: E. polecki trophozoites stained with trichrome.

Iodamoeba buetschlii

Cysts have only one nucleus with a large, usually eccentric karyosome.  They
do not have chromatoid bodies but have a compact, well defined glycogen
mass.  The cysts measure usually 10 to 12 µm (range 5 to 20 µm) and their
shape varies from ovoidal to rounded.

Line drawing of an

I. buetschlii cyst.

B C

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B, C: I. buetschlii cysts stained with trichrome.  Note the well defined glycogen
mass in B (black arrow)

Iodamoeba buetschlii have one nucleus with a large, usually central karyosome
surrounded by refractile, achromatic granules.  Their cytoplasm is coarsely
granular, vacuolated and can contain bacteria

A B

A: Line drawing of an I. buetschlii trophozoite.

B: I. buetschlii trophozoite stained with trichrome.

Iodamoeba buetschlii have one nucleus with a large, usually central karyosome
surrounded by refractile, achromatic granules.  Their cytoplasm is coarsely
granular, vacuolated and can contain bacteria, yeasts or other materials.  The
trophozoites measure usually 12 to 15 µm (range 8 to 20 µm).

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 I. buetschlii trophozoite stained

with iron hematoxylin.

Free-living Amebic infections

______________________________________
Acanthamoeba spp.
Balamuthia mandrillaris
Naegleria fowleri
Sappinia spp.

Causal Agents:

Naegleria fowleri and Acanthamoeba spp., are commonly found in lakes,

swimming pools, tap water, and heating and air conditioning units.  While only
one species of Naegleria, N. fowleri, is known to infect humans, several species
of Acanthamoeba, including A. culbertsoni, A. polyphaga, A. castellanii, A.
astronyxis, A. hatchetti, A. rhysodes, A. divionensis, A. lugdunensis, and A.
lenticulata are implicated in human disease.  An additional agent of human
disease, Balamuthia mandrillaris, is a related free-living ameba that is
morphologically similar to Acanthamoeba in tissue sections in light microscopy. 
Sappinia is a genus of free-living amebae rarely isolated from humans; cysts
and trophs have been found in the feces of many animals, including mammals
and reptiles.

Life Cycle:

Free-living amebae belonging to the genera Acanthamoeba, Balamuthia,

Naegleria and Sappinia are important causes of disease in humans and

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animals.  Naegleria fowleri produces an acute, and usually lethal, central
nervous system (CNS) disease called primary amebic meningoencephalitis
(PAM).  Acanthamoeba spp. and Balamuthia mandrillaris are opportunistic free-
living amebae capable of causing granulomatous amebic encephalitis (GAE) in
individuals with compromised immune systems.  Sappinia diploidea has been
implicated in a case of amebic encephalitis.

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Life Cycle of Acanthamoeba spp.:

Acanthamoeba spp. have been found in soil; fresh, brackish, and sea water;
sewage; swimming pools; contact lens equipment; medicinal pools; dental
treatment units; dialysis machines; heating, ventilating, and air conditioning
systems; mammalian cell cultures; vegetables; human nostrils and throats; and
human and animal brain, skin, and lung tissues.  Unlike N. fowleri,
Acanthamoeba has only two stages, cysts and trophozoites , in its life
cycle.  No flagellated stage exists as part of the life cycle.  The trophozoites
replicate by mitosis (nuclear membrane does not remain intact) .  The
trophozoites are the infective forms, although both cysts and trophozoites gain
entry into the body through various means.  Entry can occur through the eye
, the nasal passages to the lower respiratory tract , or ulcerated or broken
skin .  When Acanthamoeba spp. enters the eye it can cause severe keratitis
in otherwise healthy individuals, particularly contact lens users .  When it
enters the respiratory system or through the skin, it can invade the central
nervous system by hematogenous dissemination causing granulomatous
amebic encephalitis (GAE) or disseminated disease , or skin lesions in
individuals with compromised immune systems.  Acanthamoeba spp. cysts and
trophozoites are found in tissue.

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Life Cycle of Balamuthia mandrillaris:

Balamuthia mandrillaris has only recently been isolated from the environment
and has also been isolated from autopsy specimens of infected humans and
animals.  B. mandrillaris has only two stages, cysts and trophozoites , in its
life cycle.  No flagellated stage exists as part of the life cycle.  The trophozoites
replicate by mitosis (nuclear membrane does not remain intact) .  The
trophozoites are the infective forms, although both cysts and trophozoites gain
entry into the body through various means.  Entry can occur through the
nasal passages to the lower respiratory tract , or ulcerated or broken skin . 
When B. mandrillaris enters the respiratory system or through the skin, it can
invade the central nervous system by hematogenous dissemination causing
granulomatous amebic encephalitis (GAE) or disseminated disease , or
skin lesions in individuals who are immune competent as well as those with
compromised immune systems.  B. mandrillaris cysts and trophozoites are
found in tissue.

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Life Cycle of Naegleria fowleri:

Naegleria fowleri has three stages, cysts , trophozoites , and flagellated

forms , in its life cycle.  The trophozoites replicate by promitosis (nuclear
membrane remains intact) .  N. fowleri is found in fresh water, soil, thermal
discharges of power plants, heated swimming pools, hydrotherapy and
medicinal pools, aquariums, and sewage.  Trophozoites can turn into temporary
non-feeding flagellated forms which usually revert back to the trophozoite
stage.  Trophozoites infect humans or animals by penetrating the nasal mucosa
and migrating to the brain via the olfactory nerves causing primary amebic
meningoencephalitis (PAM).  N. fowleri trophozoites are found in cerebrospinal
fluid (CSF) and tissue, while flagellated forms are occasionally found in CSF. 
Cysts are not seen in brain tissue.

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Geographic Distribution:

While infrequent, infections appear to occur worldwide.

Clinical Features:

Acute primary amebic meningoencephalitis (PAM) is caused by Naegleria

fowleri.  It presents with severe headache and other meningeal signs, fever,
vomiting, and focal neurologic deficits, and progresses rapidly (<10 days) and
frequently to coma and death.  Acanthamoeba spp. causes mostly subacute or
chronic granulomatous amebic encephalitis (GAE), with a clinical picture of
headaches, altered mental status, and focal neurologic deficit, which
progresses over several weeks to death.  In addition, Acanthamoeba spp. can
cause granulomatous skin lesions and, more seriously, keratitis and corneal
ulcers following corneal trauma or in association with contact lens use.  Non-
contact lens users and contact lens users with safe lens care practices can
become infected.  However, poor contact lens hygiene and exposure to
contaminated water may increase the risk among contact lens users.

Laboratory Diagnosis:

In Naegleria infections, the diagnosis can be made by microscopic examination

of cerebrospinal fluid (CSF).  A wet mount may detect motile trophozoites, and
a Giemsa-stained smear will show trophozoites with typical morphology.  In
Acanthamoeba infections, the diagnosis can be made from microscopic
examination of stained smears of biopsy specimens (brain tissue, skin, cornea)
or of corneal scrapings, which may detect trophozoites and cysts.  Confocal
microscopy or cultivation of the causal organism, and its identification by direct
immunofluorescent antibody, may also prove useful.  An increasing number of
PCR-based techniques (conventional and real-time PCR) have been described
for detection and identification of free-living amebic infections in the clinical
samples listed above.  Such techniques may be available in selected reference
diagnostic laboratories.

Diagnostic findings

 Microscopy
 Molecular Methods

Treatment:
Eye and skin infections caused by Acanthamoeba spp. are generally treatable. 
Although most cases of brain (CNS) infection with Acanthamoeba have resulted
in death, patients have recovered from the infection with proper treatment. 
Amphotericin B* has been successfully used in some cases to treat PAM
caused by Naegleria fowleri.

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Microscopy

Acanthamoeba spp.

The cysts of Acanthamoeba spp. are typically 10-25 µm in diameter.  The cysts have
two walls: a wrinkled fibrous outer wall (exocyst) and an inner wall (endocyst) tha t may
be hexagonal, spherical, star-shaped or polygonal.  Cysts contain only one nucleus with
a large karyosome.  Cysts may be found in the brain, eyes, skin, lungs and other organs.

A B

A, B: Cysts of Acanthamoeba sp. from brain tissue, stained with hematoxylin and eosin
(H&E).

Trophozoites of Acanthamoeba spp. are pleomorphic and measure approximately 15-

45 µm.  They often produce many spine-like processes called acanthapodia. 
Trophozoites contain a large nucleus with a large, centrally-located karyosome but no
peripheral chromatin.  There is no flagellated trophozoite stage in Acanthamoeba spp.

C D

C: Trophozoite of Acanthamoeba sp. in tissue, stained with H&E.

D: Trophozoites of Acanthamoeba sp. in a corneal scraping, stained with H&E.

Balamuthia mandrillaris

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The cysts of Balamuthia mandrillaris are typically 10-25 µm in diameter.  The cysts
have two walls: a wrinkled fibrous outer wall (exocyst) and an inner wall (endocyst) that
may be hexagonal, spherical, star-shaped or polygonal.  Cysts contain only one
nucleus with a large karyosome.  Cysts may be found in the brain, eyes, skin, lungs and
other organs.

E F

E, F: Cysts of B. mandrillaris in brain tissue, stained with H&E. 

G H

G, H: Cysts of B. mandrillaris in brain tissue, stained with H&E. 

Trophozoites of Balamuthia mandrillaris are pleomorphic and measure approximately

15-60 µm.  They often produce long, slender pseudopodia. Trophozoites contain a
large nucleus with a large, centrally-located karyosome but no peripheral chromatin. 
There is no flagellated trophozoites stage in Naegleria spp.

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 I J

I, J: Trophozoites of B. mandrillaris in brain tissue, stained with H&E.

Naegleria fowleri

Naegleria fowleri does not form cysts in tumman tissues.  There are two forms of
trophozoites: ameboid and ameboflagellate, only the former of which is found in
humans.  The ameboid trophozoites measure 10-35 µm but when rounded are usually
10-15 µm in diameter.  In culture, trophozoites may get over 40 µm.  The cytoplasm in
granular and contains many vacuoles.  The single nucleus is large and has a large,
dense karyosome and lacks peripheral chromatin.

K L

K: Trophozoite of N. fowleri in CSF, stained with H&E.

L: Trophozoite of N. fowleri in CSF, stained with trichrome. 

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 L M

L, M: Trophozoites of N. fowleri in brain tissue, stained with H&E.

Sappinia spp.

Sappinia is a genus of free-living amebae rarely isolated from human specimens.  The
genus is found worldwide and has been isolated in the feces of many animals,
including mammals and reptiles.  Cysts and trophozoites both possess two nuclei.

N: Four trophozoites (yellow arrows) of S. diploidea in brain tissue, stained with H&E. 
In three of the amebae, the two nuclei can easily be seen.

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Indirect Immunofluorescence (IIF) assay for the detection of free-living amebic
infections.

O P

O: IIF assay for Acanthamoeba sp., 400x magnification

P: IIF assay for Balamuthia mandrillaris.

Q: IIF for Naegleria fowleri,

1000x oil magnification.

Molecular diagnosis

Real-Time PCR

A real-time PCR was developed at CDC for identification of Acanthamoeba spp.,

Naegleria fowleri, and Balamuthia mandrillaris in clinical samples.1  This assay uses
distinct primers and TaqMan probes for the simultaneous identification of these three
parasites.

33